Aspect of atherosclerosis has been hampered by a lack of complete knowledge concerning chemical and cellular aspects of lipoprotein modification. This destructive aspect is already present at the time of lesion transition from fatty streak (Type II lesion) to intermediate lesion (Type III) and then to the fibrous plaque (Type IV). In advanced atherosclerosis, the effect of tissue destruction is to weaken the arterial intima leading to plaque rupture, with consequent rapid thrombosis and often arterial occlusion causing heart attack or stroke. Future studies will continue to explore LDL oxidation chemistry in collaboration with Dr. Ned Porter. Cholesteryl arachidonate oxidation products are being defined. These products or fatty acids derived from them may directly affect cells via prostaglandin receptors and other pathways. Dr. Guyton is currently pursuing a sabbatical year working in the Neurology Division at Duke University Medical Center. Therefore, activities related to atherosclerosis have decreased GCRC Protocol 697 entitled "Atherosclerosis Studies" enabled the procurement of fasting human plasma for isolation of lipoproteins. The cellular effects and chemistry of modified lipoproteins - both oxidized and aggregated - were studied. In collaboration with Dr. Ned Porter of the Department of Chemistry we performed studies to determine the major regioisomeric hydroperoxides and alcohols formed during the early stages of LDL and HDL oxidation initiated by the thermolabile azo free radical initiator 2,2'-azobis(2-amidinopropane) dihydrochloride. After a lag time of 1 hour, significant amounts of chol-18:2-OOH and lower levels of chol-18:2-OH begin to form. At this time approximately 90% of the initial alpha-tocopherol is still present in LDL. In the early phase only 13- and 9-cis,trans-chol-18:2-OOH are formed. After depletion of antioxidants, principally alpha-tocopherol, the thermodynamic products - trans,trans-chol-18:2-OOHs - were formed in equal abundance to the cis,trans isomers. In addition to these results, this study also established HPLC methodology for the direct quantitation of specific cholesteryl ester hydroperoxides. This was published in Chemical Research in Toxicology. Subsequent studies in this area are investigating the hydroperoxides formed via oxidation of cholesteryl arachidonate in LDL and HDL. The complexity of chemical reactions possible due to the four double bonds in arachidonic raises the likelihood for formation of biological active intermediates. In atherosclerotic lesions at the critical stage of lesion transition, the most prominent biochemical manifestation is the accumulation of unesterified (free) cholesterol (see my review in Arteriosclerosis, Thrombosis, and Vascular Biology - January 1996). We asked whether hydrolysis of the ester bond might occur as cholesteryl esters in LDL are oxidized. Indeed free cholesterol content was found to increase at early stages of LDL oxidation, despite the fact that oxidation of the cholesterol ring structure would cause depletion of free cholesterol. Cholesteryl linoleate and cholesteryl palmitate labeled with tritium in the cholesterol moiety was incorporated into LDL prior to oxidation. With the linoleate, but not the palmitate, a product sequence largely comprising cholesteryl linoleate hydroperoxides, unesterified cholesterol, then unesterified oxysterols was shown to occur. Therefore, one of the chemical processes in LDL oxidation is hydrolysis of the cholesteryl ester bond. This process is probably nonenzymatic, as suggested by the lack of effect of esterase inhibitors.